Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.     

Stereotactic radiosurgery of malignant and benign intracranial lesions utilizing a patient rotator

The objective of this survey was to demonstrate whether a primary care track internal medicine residency program emphasizing community-based health care of the urban sick poor trains physicians who will continue to practice in general internal medicine or similar fields. Thirty-five primary care residents (100% of graduates) who trained from 1976 through 1993 in the Adult Primary Care Track of the Internal Medicine Residency Program at St. Vincent's Hospital, New York were used as participants.     

Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.     

Prevalence of Blastocystis hominis infection in schoolchildren from Bolivar City, Venezuela

Blastocystis hominis is found in about 25% of feces in normal asymptomatic people. Its pathogenic role is still discussed. A prospective study was performed to determine the prevalence of B. hominis infection in schoolchildren from Bolivar City. We evaluated 446 children, between five and fourteen years old, both sexes, using direct examination of feces and Willis Method. They were also evaluated clinically. Results showed that B. hominis had a prevalence of 16.8%. We did not find a statistically significant association between sex (P > 0.05) or age and infection with B. hominis (x2 = 1.94 g.l = 4). In 39 schoolchildren (52.0%) we identified other parasites along with B. hominis, the most frequent was Trichuris trichiura as helminth and Giardia lamblia as protozoan. We observed B. hominis alone in 36 cases (48.0%). There was a spectrum of clinical symptoms in 41 (54.7%) of all children evaluated. Diarrhea was the most frequently clinical manifestation observed. Other studies are necessary to determine clinical relevance of B. hominis in school population in Bolivar City.     

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.     

The CD40-CD154 system in anti-infective host defense

Research in the past few years has documented significant advances in our understanding of the CD40-CD40 ligand (CD154) system in diverse immune functions. This system influences many T cell mediated inflammatory immune responses and effector functions, unmasking a previously unexpected role for CD40-CD154 in cell mediated immunity. Manipulation of CD154 in animal models of infection by the use of CD154-deficient mice or anti-CD154 antibodies has shown the importance of this system in the initiation of the inflammatory response, in the activation of antigen-presenting cells and in resistance to infections.     

Investigation of I1-imidazoline receptors using microphysiometry and molecular modelling

A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.     

Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium

We set out to validate the concept of three-dimensional (3D) angiography. We evaluated the sensitivity and the quality of morphological analysis mode possible by an experimental system for imaging cerebrovascular disease versus standard digital subtraction angiography (DSA). The system, the 3D Morphometer, is a computerised X-ray angiography unit capable of acquiring a set of two-dimensional (2D) projections during a rotation and then reconstructing a 3D volume from them. We studied 78 patients with suspected cerebrovascular disease. 3D and 2D images (standard 2D DSA performed during the same procedure), were reviewed blindly to assess detection and display of morphological characteristics of cerebrovascular diseases. We found 53 aneurysms, 22 arteriovenous malformations and two venous angiomas. On 3D angiography we detected two aneurysms we missed on 2D angiography. In 47 aneurysms on which further data were obtained during surgery or embolisation, the 3D angiography allowed more accurate analysis of the neck and surrounding vessels in cases in which the 2D angiographic findings were doubtful. Assessment of arteriovenous malformations was equivalent with both techniques. Under the conditions of our study, the technical constraints being the same for both methods, 3D angiography was superior to 2D angiography. Implementation on C-arm vascular systems is being evaluated.     

Pharmacokinetic-pharmacodynamic modeling of the anticonvulsant and electroencephalogram effects of phenytoin in rats

In this study a pharmacokinetic-pharmacodynamic model is proposed for drugs with nonlinear elimination kinetics. We applied such an integrated approach to characterize the pharmacokinetic-pharmacodynamic relationship of phenytoin. In parallel, the anticonvulsant effect and the electroencephalogram (EEG) effect were used to determine the pharmacodynamics. Male Wistar-derived rats received a single intravenous dose of 40 mg . kg-1 phenytoin. The increase in the threshold for generalized seizure activity (TGS) was used as the anticonvulsant effect and the increase in the total number of waves in the 11.5 to 30 Hz frequency band was taken as the EEG effect measure. Phenytoin pharmacokinetics was described by a saturation kinetics model with Michaelis-Menten elimination. Vmax and Km values were, respectively, 386 +/- 31 microg . min-1 and 15.4 +/- 2.2 microg . ml-1 for the anticonvulsant effect in the cortical stimulation model and 272 +/- 31 microg . min-1 and 5.9 +/- 0.7 microg . ml-1 for the EEG effect. In both groups, a delay to the onset of the effect was observed relative to plasma concentrations. The relationship between phenytoin plasma concentrations and effect site was estimated by an equilibration kinetics routine, yielding mean ke0 values of 0.108 and 0.077 min-1 for the anticonvulsant and EEG effects, respectively. The EEG changes in the total number of waves could be fitted by the sigmoid Emax model, but Emax values could not be estimated for the nonlinear relationship between concentration and the increase in TGS. An exponential equation (E = E0 + Bn . Cn) derived from the sigmoid Emax model was applied to describe the concentration-anticonvulsant effect relationship, under the assumption that Emax values cannot be reached within acceptable electric stimulation levels. This approach yielded a coefficient (B) of 2.0 +/- 0.4 microA . ml . microg-1 and an exponent (n) of 2.7 +/- 0.9. The derived EC50 value of 12.5 +/- 1. 3 microg . ml-1 for the EEG effect coincides with the "therapeutic range" in humans.